Method for the production of collagen: collagen produced through the method and use of collagen

ABSTRACT

The present invention relates to a process for the production of collagen from animal intestines etc. It is characterized in that the starting material is mixed with ice-water at a pH of 5,5, that the mixture is disintegrated and then heated to 40°-42° C. and hydrolysed at a pH of 10,5 using a proteolytic enzyme. After finishing of the hydrolyses pH is regulated to 5,5 and the collagen is separated and collected. The invention also encloses collagen produced through the process and a number of new application areas for collagen.

TECHNICAL FIELD

The present invention relates to an improved way for the production ofcollagen and collagen produced through the process and the use ofcollagen in different areas.

PRIOR ART

Collagen is a fibre formed substance consisting of 18 amino-acids and itprovides the main part of the intestines and stomach of animals but isalso included in other parts of the animal body, for example in theinterial part of the skin in the shape of hide, in lunges and uddersetc. One has earlier primarily used the collagen in its native shape,mostly as sausage skin made from cleaned intestines. During resent yearsone has also produced collagen in its clean shape starting from theabove said body parts of animals and used this clean collagen fordifferent purposes such as artificially made sausage skins, additionalagents for treatment articles, surgery threads etc. A known way ofproducing collagen is described in the Swedish Patent No. 8003876-3 inwhich a process is described that comprises cutting cleaned intestinesetc. in stripes and pieces which are deep frozen and ground where uponthey are subjected to the influence of an enzyme in a water solutionwhereby the aminoacids which do not provide a part of the collagen ishydrolysed so that insoluble remaining collagen can be removed and usedafter further treatment. Another process for producing collagencomprises the use of lime for separating the collagen fibrils. Thisprocess is time consuming and results also in that free collagenmolecules are separated which impairs the properties of the end product.

TECHNICAL PROBLEM

It has since long been a desire to bring about a process which is betterthan the two above mentioned processes for the production of collagen.Thus, the first of said above mentioned processes includes that one hasto freeze this disintegrated starting materials which iscircumstantially and expensive and that one has to carry out thehydrolysis at a relatively low pH, approximately 8, using a relativelyweek enzyme during a period of 2-5 hours. This process is both costlyand time consuming.

By the second of the above mentioned processes lime has to be addedwhich results in a lower grade end product at the same time as anextended process will be necessary.

THE SOLUTION

By the present invention one has solved the problems connected with theabove mentioned processes and brought about a process for the productionof collagen from by-products of slaughtered and cut animals, such asintestines, stomach etc. which is characterized by the following processsteps:

a) the starting materials are cleaned and immersed in ice-water, pHbeing regulated to approximately 5,5,

b) the mixture of starting materials and ice-water is ground whereuponfurther water is added so that the ground mixture contains approximatelyequal weight parts of starting materials and water,

c) the mixture is heated to 40°-42 ° C. and pH is regulated to at most11, preferably 10,5 whereupon a proteolytic enzyme, for instance alkalasin an amount corresponding to 60 Anson-units per kilo solid substance isadded so that hydrolysis of other proteins than collagen is carried outduring maintaining the pH value by adding alkali until the hydrolyses iscompleted and alkali is no more consumed,

d) pH is regulated to 5,5 by adding an acid whereupon

e) separated collagen is collected.

According to the invention it is suitable that pH in steps a) and d) isregulated by means of hydrochloric acid, citric acid or lactic acid.

The hydrolysing is carried out according to the invention during aperiod of time of 13/4 hours to 31/2 hours while adding sodium hydroxideas alkali.

According to the invention the separated collagen can be collected bysifting, cleaning with water at about 40° C. and centrifugation ordecanting. Only one wash with water is sufficient.

It is according to the invention further suitable that the collagen ishomogenized after being collected at pH c:a 3 by alternatively addingacids and water while mechanically treating until the desired endconcentration of collagen is obtained.

It is according to the invention suitable for obtaining a cleartransparent film from the completed collagen that it is mixed withreduction agents, such as ascorbic acid and sodium sulphite in an amountof up to 2 weight per cent, and cross-binding agents for exampleglutaraldehyde in an amount of approximately 0,1 weight per cent and5-10 weight per cent glycerol as softener, all calculated on solidcollagen.

The invention comprises also collagen produced by the process accordingto the above description and use of collagen as a binder for meatproducts, as a film for packing of food and medicines, as a carrier bythe production of colours, as artificial leather, as a gel for treatmentof wounds, as a cover on chips for electronic purposes and as coverswithin the paper industry.

The raw materials that can be used in the process according to theinvention is primarily such materials that come from the digestingorgans of the animals such as rumen, fourth stomach, second stomach,intestines and lunges and udder from ruminants. Also the large intestinefrom pigs can be used.

When using an intestine this is first emptied by having a machinesqueezing out the content and the inner part of the intestines calledthe mucosa by squeezing between rubber rollers. Any limitation of theassortment of raw materials does not exist and all such that comprisecollagen in sufficient amount can be used. The raw materials arepretreated advantageously by removing fat, the so called mesentery andthe like.

The cleaned starting materials are then mixed with ice-water whichsuitably have an addition of acetic acid so that a pH of about, 5,5 isobtained. Herethrough the intestine is rapidly cooled and possiblebacteria evolution will cease. One avoids to freeze the startingmaterials that can be transported and maintained in this conditionbefore they are ground. This is important as the production of collagenonly occurs at very few places and the starting materials have to betransported from the slaughter-houses to the collagen-factory.

At the collagen-factory pure water is added so that the startingmaterials are present in an amount of about 50 per cent by weight of themixture. This is heated to 40°-42° C. when stirring and one adds sodiumhydroxide so that pH is raised to 10,5 and the temperature adjusted to40°-42° C. It is important that the temperature does not exceed thisvalue as the collagen than will undergo crystalline changes and at stillhigher temperatures will change into gelatine. At the desiredtemperature and pH value the proteolytic enzymes are added whichpreferably consist of alkalas. At this high pH a strong concentration ofenzymes can be added and the time for hydrolysing is therefore short,from 11/2 hour to 31/2 hours. During the hydrolyses the alkali isconsumed so that alkali has to be added continuously maintaining a pH of10,5. When the hydrolyses is completed, which is noticed by the factthat the pH does not sink any more when alkali is not added, the pH ofthe mixture is lowered to 5,5 by the addition of an acid, for examplehydrochloric acid, acetic acid, citric acid or lactic acid. What occursduring the hydrolyses is that the alkalas is hydrolysing the proteinswhich are not a part of the collagen.

At pH 5,5 lumps are formed and a contraction of the collagen occurs.This depends on that the isoelectric point of the collagen is at pHabout 5,5, which is generally known. In this shape it is preferable toseparate the collagen suitably through a sift or the like.

The collagen is finally washed in water at a temperature of about 40° C.and is centrifugated or decanted. At the centrifugation the collagennormally gets a solids content of about 22 per cent and at thedecantation about 30 per cent.

To use the collagen for the intended purpose it is preferable that it ishomogenized which occurs through mechanical treatment in a vacuum mixersuitably with a dough hook or the like when acid such as hydrochloricacid or lactic acid is added together with water so that pH 3 and adesired concentration of collagen is obtained. After the homogenisationthe collagen is set to ripen at a low temperature, normally 8°-10° C.during a period of time of 24 hours.

When the collagen shall be used for film forming, for example film inthe shape of eatable sausage skin, a cross-linking agent for exampleglutaric aldehyde is added to the collagen in an amount of about 0,1weight per cent and a softener in the shape of glycerol in an amount of5-10 weight per cent. To obtain a film which is clear and transparentalso an anti-oxidant such as ascorbic acid or sodium-bisulphate in anamount of up to 2 per cent by weight, preferably 0,02-0,1 per cent byweight should be added.

As a by-product from the collagen production animal pellets can beproduced from the hydrolysing water by evaporation while addingvitamins, minerals, and taste agents or it can be used as wet fodderwhich then must be consumed rapidly so that putrefaction does not occur.

The proteolytic enzyme used consists preferably of Novos alkalas 2,6which contains 60 Anson units per 25 millilitre. This alkalas is about 4times stronger than earlier known varieties of alkalas. Together withthe high pH-value during the hydrolyses it therefore gives a very rapidhydrolyses reaction, which in its turn means an economic advantage. Thisprocess also gives a product having a small fat content, which is ofgreat importance for the succeeding film formation.

EXAMPLE:

6,5 kilogram cleaned pig intestines are immersed in ice-water having apH of 5,5. The pH-value is regulated by means of acetic acid. Theintestines and the ice-water are ground whereupon water is added so thata total amount of water plus intestines became 13 kilogram, that is 6,5kilogram water and 6,5 kilogram cleaned pig intestines.

The mixture is stirred and heated to 40°-41° C. during addition of about143 millilitre 4M NaOH until a pH of 10,5 was obtained. Thereafter aproteolytic enzyme in the shape of alkalas 2,6L from Novo in an amountof about 150 millilitre was added. pH was maintained constant at 10,5during about 11/2 hour at further addition of 4M NaOH so that the totalamount NaOH became 253 millilitre.

After the hydrolysing the pH was lowered to 5,5 by the addition ofhydrochloric acid. The collagen produced contracted and formed lumps.

The collagen was separated over a sift and washed in water at atemperature of 40° C. and a water amount of 6-7 kilogram duringstirring. The washing time was 20-25 minutes. Collagen was thencollected by centrifugation and was homogenized in a vacuum mixer with asuitable dough hook. During the homogenisation water and hydrochloricacid was added so that a pH of 3 and a solids content of 8 per cent wasobtained. During the homogenisation the temperature of the collagen wasnot higher than 15°-18° C.

After the homogenisation the collagen was ripened during a period oftime of 24 hours and at a temperature of 8°-10° C. whereupon it wassifted for removing of possible lumps or impurities in a special siftunder pressure.

After addition of ascorbic acid, glutaric aldehyde and glycerol thecollagen was extruded to a clear film.

By the present invention one has accordingly brought about an economicprocess which results in collagen having a very high quality and whichcan be used in a number of new areas such as declared above.

I claim:
 1. A process for separating collagen from animal tissuecomprising the steps of:immersing said collagen containing tissue in amixture of frozen and liquid water, said mixture having a pH which ismaintained at about 5.5; heating said collagen and water mixture to atemperature no higher than about 42 C while regulating the pH of saidcollagen and water mixture such that it does not exceed about 11; addingto said heated collagen and water mixture an amount of at least oneproteolytic enzyme which is sufficient to hydrolyze protein in saidtissue, other than said collagen; hydrolyzing said protein; adjustingthe pH of the collagen and water mixture to about 5.5; and separatingsaid collagen from said mixture.
 2. The process of claim 1, furthercomprising the step of adding additional water to said collagen andwater mixture until approximately 50% of the mixture by weight istissue.
 3. The process of claim 1 further comprising the step of saidcollagen and grinding said tissue prior to said heating step.
 4. Theprocess of claim 1 wherein, during said heating step, the pH ismaintained at no more than about 10.5.
 5. The process of claim 1wherein, during said hydrolyzing step, the pH is maintained at no morethan about 10.5.
 6. The process of claim 1 wherein the amount ofproteolytic enzyme added is about 60 Anson units per kilogram of solid.7. The process of claim 1 further comprising the step of cleaning saidtissue prior to said immersion step.
 8. A process for separatingcollagen from animal tissue comprising the steps of:providing cleancollagen containing tissues; immersing said collagen containing tissuein a mixture of frozen and liquid water, said mixture of frozen andliquid water not exceeding the weight of the collagen containing tissueadded, while maintaining the pH of said mixture at about 5.5; grindingsaid collagen and water mixture; adding additional water if necessary toprovide a mixture which is approximately 50% by weight water and 50% byweight collagen; heating said collagen and water mixture to atemperature no higher than about 42 C while regulating the pH such thatit is no higher than about 11; adding to said heated collagen and watermixture an amount of a proteolytic enzyme which is sufficient tohydrolyze protein in said tissue other than said collagen andhydrolyzing said protein while maintaining said pH of said mixture;lowering the pH of said mixture to about 5.5 following substantialcompletion of the hydrolysis reaction; and separating said collagen fromsaid mixture.
 9. The process according to claims 1 or 8 wherein an acidselected from the group consisting of hydrochloric acid, citric acid orlactic acid is used to maintain said pH of said mixture at about 5.5.10. The process of claims 1 or 8 where hydrolysis is carried out for aperiod of between 1.75 and 3.5 hours.
 11. The process of claims 1 or 8wherein said pH of said mixture is maintained at a level of at most 11during said heating and hydrolysis step by the addition of sodiumhydroxide.
 12. The process of claims 1 or 8 wherein said collagen iscollected by fixing and washing said collagen with water at about 40 Cfollowed by centrifugation or decantation.
 13. The process according toclaim 12 wherein said collagen is homogenized after collection at a pHof about
 3. 14. The process of claims 1 or 8 further comprising thesteps of producing a clear transparent film by mixing the collectedcollagen with a reducing agent in the amount of about 2 weight percent,and a cross-linking agent in the amount of about 0.1 weight percent anda softener in an amount of about 5-10 weight percent based on saidcollagen, and extruding said film.